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A role for nucleotides in support of breast cancer angiogenesis: heterologous receptor signalling.

Yokdang N, Tellez JD, Tian H, Norvell J, Barsky SH, Valencik M, Buxton IL - Br. J. Cancer (2011)

Bottom Line: We examined sNDPK secretion and its effects on human endothelial cells.Nucleoside diphosphate kinase-B stimulates cell growth and migration in a concentration-dependent manner comparable to the effect of vascular endothelial growth factor.Understanding the mechanism of action of sNDPK and P2Y(1) nucleotide signalling in metastasis and angiogenesis represent new therapeutic targets for anti-angiogenic therapies to benefit patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Centre for Molecular Medicine, University of Nevada School of Medicine, Mail Stop 573, Reno, NV 89557, USA.

ABSTRACT

Background: Human breast carcinoma cells secrete an adenosine 5'-diphosphate transphosphorylase (sNDPK) known to induce endothelial cell tubulogenesis in a P2Y receptor-dependent manner. We examined sNDPK secretion and its effects on human endothelial cells.

Methods: Nucleoside diphosphate kinase (NDPK) secretion was measured by western blot and enzyme-linked immunosorbent assay, while transphosphorylase activity was measured using the luciferin-luciferase ATP assay. Activation of MAPK was determined by western blot analysis, immunofluorescence and endothelial cell proliferation and migration.

Results: A panel of breast cancer cell lines with origin as ductal carcinoma, adenocarcinoma or medullary carcinoma, secrete sNDPK-A/B. Addition of purified NDPK-B to endothelial cultures activated VEGFR-2 and Erk(1/2), both of which were blocked by inhibitors of NDPK and P2Y receptors. Activation of VEGFR-2 and ErK(1/2) by 2-methylthio-ATP (2MeS-ATP) was blocked by pretreatment with the P2Y(1)-specific antagonist MRS2179, the proto-oncogene non-receptor tyrosine kinase (Src) inhibitor PP2 or the VEGFR-2 antagonist SU1498. Nucleoside diphosphate kinase-B stimulates cell growth and migration in a concentration-dependent manner comparable to the effect of vascular endothelial growth factor. Treatment of endothelial cells with either NDPK-B or 2MeS-ATP induced migration, blocked by P2Y(1), Src or VEGFR-2 antagonists.

Conclusion: sNDPK supports angiogenesis. Understanding the mechanism of action of sNDPK and P2Y(1) nucleotide signalling in metastasis and angiogenesis represent new therapeutic targets for anti-angiogenic therapies to benefit patients.

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Related in: MedlinePlus

Antagonism of 2MeS-ATP or NDPK-B-mediated HEC migration is consistent with Src activation of VEGFR-2. HEC were seeded on the upper surface of collagen-coated membranes as described in the text. (A) Ten units of NDPK-B added in the presence of phosphoryl donor and acceptor that produced Vmax conditions for ATP production. (B). All antagonist treatments (A and B) produced highly significant blockade. (C) Micrographs representative of the result in each experiment. Membranes were prepared and stained as described; migrated cells were counted and values expressed as percentage of the maximum stimulation defined as VEGF-stimulated migration. Values are mean±s.e.m., n=3–5 and all statistical comparisons of the effects of inhibitors on stimulated migration (A and B) are significant at P<0.001.
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fig7: Antagonism of 2MeS-ATP or NDPK-B-mediated HEC migration is consistent with Src activation of VEGFR-2. HEC were seeded on the upper surface of collagen-coated membranes as described in the text. (A) Ten units of NDPK-B added in the presence of phosphoryl donor and acceptor that produced Vmax conditions for ATP production. (B). All antagonist treatments (A and B) produced highly significant blockade. (C) Micrographs representative of the result in each experiment. Membranes were prepared and stained as described; migrated cells were counted and values expressed as percentage of the maximum stimulation defined as VEGF-stimulated migration. Values are mean±s.e.m., n=3–5 and all statistical comparisons of the effects of inhibitors on stimulated migration (A and B) are significant at P<0.001.

Mentions: We determined the effect of extracellular NDPK-B vs specific P2Y1 activation by 2MeS-ATP on endothelial cell migration using a modified Boyden chamber assay. Activation of P2Y1R by 2MeS-ATP or extracellular NDPK-B is as effective as VEGF in stimulating endothelial cell migration (Figure 7C). Stimulation of HEC by either 10 μ 2MeS-ATP or 10 units of NDPK-B in the presence of GTP and ADP induced similar levels of migration (∼7-fold above the negative control, Figure 7). Human cord blood endothelial colony forming cells migration stimulated by 2MeS-ATP or NDPK-B plus GTP/ADP was significantly reduced by the P2Y1R antagonist MRS2179 or the Src inhibitor PP2 and to the same extent, while the VEGFR-2 antagonist SU1498 (Figure 7) was far more effective blocking essentially all of the stimulation produced by either agonist.


A role for nucleotides in support of breast cancer angiogenesis: heterologous receptor signalling.

Yokdang N, Tellez JD, Tian H, Norvell J, Barsky SH, Valencik M, Buxton IL - Br. J. Cancer (2011)

Antagonism of 2MeS-ATP or NDPK-B-mediated HEC migration is consistent with Src activation of VEGFR-2. HEC were seeded on the upper surface of collagen-coated membranes as described in the text. (A) Ten units of NDPK-B added in the presence of phosphoryl donor and acceptor that produced Vmax conditions for ATP production. (B). All antagonist treatments (A and B) produced highly significant blockade. (C) Micrographs representative of the result in each experiment. Membranes were prepared and stained as described; migrated cells were counted and values expressed as percentage of the maximum stimulation defined as VEGF-stimulated migration. Values are mean±s.e.m., n=3–5 and all statistical comparisons of the effects of inhibitors on stimulated migration (A and B) are significant at P<0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101911&req=5

fig7: Antagonism of 2MeS-ATP or NDPK-B-mediated HEC migration is consistent with Src activation of VEGFR-2. HEC were seeded on the upper surface of collagen-coated membranes as described in the text. (A) Ten units of NDPK-B added in the presence of phosphoryl donor and acceptor that produced Vmax conditions for ATP production. (B). All antagonist treatments (A and B) produced highly significant blockade. (C) Micrographs representative of the result in each experiment. Membranes were prepared and stained as described; migrated cells were counted and values expressed as percentage of the maximum stimulation defined as VEGF-stimulated migration. Values are mean±s.e.m., n=3–5 and all statistical comparisons of the effects of inhibitors on stimulated migration (A and B) are significant at P<0.001.
Mentions: We determined the effect of extracellular NDPK-B vs specific P2Y1 activation by 2MeS-ATP on endothelial cell migration using a modified Boyden chamber assay. Activation of P2Y1R by 2MeS-ATP or extracellular NDPK-B is as effective as VEGF in stimulating endothelial cell migration (Figure 7C). Stimulation of HEC by either 10 μ 2MeS-ATP or 10 units of NDPK-B in the presence of GTP and ADP induced similar levels of migration (∼7-fold above the negative control, Figure 7). Human cord blood endothelial colony forming cells migration stimulated by 2MeS-ATP or NDPK-B plus GTP/ADP was significantly reduced by the P2Y1R antagonist MRS2179 or the Src inhibitor PP2 and to the same extent, while the VEGFR-2 antagonist SU1498 (Figure 7) was far more effective blocking essentially all of the stimulation produced by either agonist.

Bottom Line: We examined sNDPK secretion and its effects on human endothelial cells.Nucleoside diphosphate kinase-B stimulates cell growth and migration in a concentration-dependent manner comparable to the effect of vascular endothelial growth factor.Understanding the mechanism of action of sNDPK and P2Y(1) nucleotide signalling in metastasis and angiogenesis represent new therapeutic targets for anti-angiogenic therapies to benefit patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Centre for Molecular Medicine, University of Nevada School of Medicine, Mail Stop 573, Reno, NV 89557, USA.

ABSTRACT

Background: Human breast carcinoma cells secrete an adenosine 5'-diphosphate transphosphorylase (sNDPK) known to induce endothelial cell tubulogenesis in a P2Y receptor-dependent manner. We examined sNDPK secretion and its effects on human endothelial cells.

Methods: Nucleoside diphosphate kinase (NDPK) secretion was measured by western blot and enzyme-linked immunosorbent assay, while transphosphorylase activity was measured using the luciferin-luciferase ATP assay. Activation of MAPK was determined by western blot analysis, immunofluorescence and endothelial cell proliferation and migration.

Results: A panel of breast cancer cell lines with origin as ductal carcinoma, adenocarcinoma or medullary carcinoma, secrete sNDPK-A/B. Addition of purified NDPK-B to endothelial cultures activated VEGFR-2 and Erk(1/2), both of which were blocked by inhibitors of NDPK and P2Y receptors. Activation of VEGFR-2 and ErK(1/2) by 2-methylthio-ATP (2MeS-ATP) was blocked by pretreatment with the P2Y(1)-specific antagonist MRS2179, the proto-oncogene non-receptor tyrosine kinase (Src) inhibitor PP2 or the VEGFR-2 antagonist SU1498. Nucleoside diphosphate kinase-B stimulates cell growth and migration in a concentration-dependent manner comparable to the effect of vascular endothelial growth factor. Treatment of endothelial cells with either NDPK-B or 2MeS-ATP induced migration, blocked by P2Y(1), Src or VEGFR-2 antagonists.

Conclusion: sNDPK supports angiogenesis. Understanding the mechanism of action of sNDPK and P2Y(1) nucleotide signalling in metastasis and angiogenesis represent new therapeutic targets for anti-angiogenic therapies to benefit patients.

Show MeSH
Related in: MedlinePlus